SiRNA
Small interfering RNA (siRNA) is a powerful tool used in the study of protein function. siRNA is used to selectively inhibit gene expression by preventing translation of mRNA into protein. When using a chemical inhibitor to prevent protein function there is a chance that there will be unintended interactions with proteins other than the target protein. Using siRNA to ‘knock down’ protein expression is more selective than chemical inhibitors because it is able to target single proteins and reduce the possibility of non-selective protein inhibition. This is especially important in studying the interactions or role of a protein in a signaling pathway to ensure only one protein is being manipulated at a given time. siRNAs were first characterized in 1993 when scientists researching C. elegans observed a short dsRNA sequence was able to bind to a larger mRNA strand with a complementary sequence and inhibit translation. http://www.ncbi.nlm.nih.gov/pubmed/8252621 Structure/Function siRNA strands average about 20-25 base pairs of double stranded RNA that contain a sequence that is complementary to a region in a strand of mRNA. Today siRNA can be created synthetically so specific sequences can be designed for any gene of interest. The siRNA is incorporated into the cell using a process called a transfection. Common methods of transfection use chemicals or electroporation in order to permeabilize the membrane and allow for the siRNA to enter the cells cytoplasm http://en.wikipedia.org/wiki/SiRNA . As a control to ensure the transfection process does not have an effect on gene expression or protein function, cells are transfected with a nonsense strand of siRNA that will not interact with any mRNA. Once in the cell, siRNA is processed by the enzyme Dicer, which is an endonuclease that allows for the siRNA to interact with the RNA-induced silencing complex (RISC) http://www.nature.com/scitable/topicpage/small-non-coding-rna-and-gene-expression-1078 . This complex of proteins unwinds the double stranded siRNA and allows for it to bind to it complementary mRNA sequence. After the siRNA has bound to its complementary mRNA, the double stranded mRNA is recognized by the cell and is degraded. This allows for gene transcription to continue to function normally while still knocking down protein expression. One drawback of using siRNA is that its effects are transient and can be lost over time, which can be a problem if cells are cultured for a long period. One method around this is to stably transfect cells with an expression vector containing a short hairpin RNA (shRNA) that is continuously replicated within the cell. The shRNA then interacts like the siRNA by binding to mRNA leading to its degradation. Current Research In the study outlined below siRNA was used to knock down expression of the membrane protein P2Y2R. P2Y2R is a G-coupled protein receptor that to activation of EGFR in an ATP-dependent manner. By knocking down expression of P2Y2R, cells no longer activate and EGFR in response to ATP. This was important in showing that the P2Y2 receptor is mediates EGFR activation in response to ATP, which is a key factor in wound response and cell migration. Using siRNA researchers were able to better illustrate the pathway outlined in the figure that is involved in the wound response. siRNAs for other genes involved were also used to help identify their roles in the pathway outlined http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0054391 References 1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 Dec 3;75(5):843-54. 2 http://en.wikipedia.org/wiki/SiRNA 3 Phillips, T. (2008) Small non-coding RNA and gene expression. Nature Education 1(1):115 4 Sham D, Wesley UV, Hristova M, van der Vliet A (2013) ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17. PLoS ONE 8(1): e54391. doi:10.1371/journal.pone.0054391